Tibet minipigs at the age of 6 months were obtained from the Institute of Comparative Medicine and Laboratory Animal Center of Southern Medical University (Guangzhou, Guangdong, China). The animals were housed under standard laboratory conditions and given food and water ad libitum by Southern Medical University Laboratory Animal Center. All experiments using experimental minipigs were approved by the Animal Care and Use Committee of the Animal Center of Southern Medical University (Animal Welfare Assurance L2016088).
Isolation and cultivation of bone marrow-derived MSCs from Tibet minipigs
Primary pMSCs were isolated from the bone marrow of Tibet minipigs as previously described by Lee et al.  and Soleimani et al. . All procedures were performed under full anesthesia and aseptic conditions. After sterilization, bone marrow was extracted from the iliac crest bone by a bone marrow extractor in a noninvasive manner and was carefully pipetted onto Ficoll-Paque solution (GE Healthcare, Piscataway, NJ, USA) at a ratio of 4:3 without mixing. The mixture was centrifuged at 500 rpm for 10 min at room temperature, and pMSCs were collected from the interface layer.
Cells were plated into a series of 25 cm2 flasks at a density of 7.5 × 105 cells per flask in Dulbecco’s modified Eagle’s medium (DMEM; Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Paisley, UK), 10 ng/ml basic fibroblast growth factor (bFGF; Sigma-Aldrich, St. Louis, MO), 1.0% Glutamax (Gibco, Shanghai, China), and 1.0% penicillin–streptomycin (Gibco). Half of the medium was replaced with fresh medium, and non-adherent cells were carefully removed after 4 h. Thereafter, half of the medium was changed every 8 h for up to 3 days after the initial culture. The adherent fibroblast-like and spindle-shaped cells were washed with phosphate-buffered saline (PBS) and replaced with fresh medium every 2 days.
Flow cytometric analysis
The MSC-like cells were harvested by trypsinization (Gibco) and incubated with 1% bovine serum albumin (BSA) for 1 h at 4 °C to block nonspecific Fc-mediated interactions, then incubated in the dark at 4 °C for 30 min with 400 μl of CD45-FITC (BD Biosciences, 340664, Franklin Lakes, NJ), CD34-APC (BD, 555824), CD105-FITC (BD, 561443), CD73-PE (BD, 561258), and CD90-APC (BD, 559869) antibodies. The cells were stained with PE- or FITC-labeled IgG as an isotype control. The cells were evaluated by a FACSCalibur flow cytometer (Becton–Dickinson, San Jose, CA) and analyzed with FlowJo software (Tree Star Inc., Ashland, Oregon, USA). The percentage of stained cells was calculated relative to the isotype control.
In vitro differentiation ability was examined in mesenchymal lineages (osteocytes, adipocytes, and chondrocytes). For osteogenesis, pMSCs were seeded into 6-well plates at a density of 2 × 106 cells per well. When the cells reached 80% confluence, the cell culture medium was supplemented with osteogenic differentiation medium (Cyagen Biosciences, Guangzhou, China) containing 10% FBS, 10 mM β-glycerophosphate, 0.1 mM ascorbic acid, and 10 nM dexamethasone in DMEM. The medium was replaced every 3 days for 1 week. Then, the cells were stained with 1% alizarin red S and analyzed under an inverted microscope (Nikon Eclipse Ti-S inverted microscope, USA).
For adipogenesis, pMSCs were seeded into 6-well plates at a density of 2 × 106 cells per well. The cells were treated with adipogenic differentiation induction medium (Cyagen) containing 10 mg/ml insulin, 100 mM indomethacin, 500 mM 3-isobutyl-1-methyl xanthine, and 1 mM dexamethasone in DMEM for 3 days, and the medium was replaced with adipogenic differentiation maintenance medium (Cyagen) containing 10% FBS and 10 mg/ml insulin in DMEM for another 24 h. After 5 induction-maintenance cycles, the culture conditions were changed to adipogenic differentiation maintenance medium with replacement every 3 days. After 7 days, the cells were stained with oil red O according to standard protocols (Cyagen) and analyzed under an inverted microscope (Nikon).
For chondrogenesis, pMSCs were supplemented with chondrogenic differentiation medium (Cyagen) containing 10 ng/ml transforming growth factor (TGF)-β3, 40 mg/ml proline, 100 mg/ml pyruvate, 10−7 M dexamethasone, a 1:100 dilution of ITS + Premix, and 50 mg/ml ascorbate-2-phosphate, which was replaced every 3 days. After 2–3 weeks of induction, the cells were stained with Alcian blue and subjected to histological analysis.
Human cDNA for Zbtb48 was purchased from Vigene Biosciences (Vigene Biosciences, NM0_005341, Shandong, China) and cloned into the pCDH-GFP-puro lentiviral vector to generate the pCDH-TZAP-GFP-puro recombination plasmid. The other plasmids used in this study were lentiCRISPRV2 (Addgene, Plasmid 52961), psPAX2 (Addgene, Plasmid 12260) and pMD2G (Addgene, Plasmid 12259).
pCDH-TZAP-GFP-puro or the pCDH-GFP-puro control plasmid was transfected into HEK293T cells with the packaging plasmids psPAX2 and pMD2G using Lipofectamine 2000 (Invitrogen). The culture supernatant was collected after 72 h, and the harvested lentiviruses were used to infect target pMSC cells. For lentiviral infection, pMSCs were seeded into 6-well plates and grown to 80% confluence, followed by incubation with the LV-TZAP or LV-GFP virus for 24 h and selection with 2.5 μg/ml puromycin for 3 days.
TZAP knockout cells were generated by using lentiviral CRISPR/Cas9 to facilitate specific genome editing in pMSCs. The target sequence of the TZAP sgRNA was designed through the CRISPR Design Tool (http://crispor.tefor.net/). Targeting of TZAP in pMSCs was carried out with the following sgRNA: TGCGATGCCACCTTGGACGT (Exon 2). The synthetic gRNA oligos were annealed following a standard protocol, cloned into the vector ‘LentiCRISPR v2’ at the BsmBI restriction site and confirmed by sequencing . Then, the recombination plasmid was cotransfected into HEK293T cells with the packaging plasmids psPAX2 and pMD2G to produce lentivirus. The lentivirus was used to infect the target cells at a multiplicity of infection (MOI) of 10. LentiCRISPRv2 without gRNA was used as a control. Antibiotic selection was performed for 3 days after transduction.
Primers were designed for the pig TZAP gene. Genomic DNA was isolated using a TIANamp Genomic DNA Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. The primers used to detect TZAP mutations were as follows: TZAP-F: 5′-AGGCTTTCTCTTGCTACC-3′ and TZAP-R: 5′-CAATCCTCAGGCTCCTTG-3′. The PCR products were then gel-purified and sequenced to confirm successful gene editing. The efficiency and spectra of the mutations were analyzed by TIDE software .
Cell counting kit-8 (CCK8) assay
To quantitatively determine cell proliferation, pMSCs were seeded into 96-well tissue culture plates at a density of 2500 cells per well in complete culture medium. Three replicates were set up for the test. The number of cells was estimated using a CCK8 assay (Bestbio, Shanghai, China) at each time point (24, 48, 72, 96, and 120 h) according to the manufacturer’s instructions. Then, 10 μl of CCK8 was added to each well and incubated for another 2 h. Optical density values (OD) were measured at a 450 nm wavelength on a microplate reader (Molecular Devices, Sunnyvale, CA, USA).
5-Ethynyl-2′-deoxyuridine cell proliferation assay (EdU assay)
The assay was performed by using a Cell-Light™ EdU Apollo 567 In Vitro Imaging Kit (EdU; Guangzhou RiboBio, Guangzhou, China) as described previously . Briefly, 5000 pMSCs were plated into 96-well tissue culture plates for 48 h, and the cells were exposed to EdU at a concentration of 50 μM and incubated for 2 h, followed by fixation with 4% formaldehyde for 30 min.
After fixation, the cells were stained with the staining mixture for 30 min and then washed twice with PBS/0.5% Triton X-100. Finally, the cells were counterstained with Hoechst 33342 and imaged by fluorescence microscopy (Nikon Eclipse Ti-S inverted microscope, USA).
Staining of SA-β-gal was performed by using the Senescence β-Galactosidase Staining Kit (Cell Signaling Technology, Danvers, MA) according to the manufacturer’s specifications. pMSCs were plated into a 96-well plate and fixed with 1× fixative solution. After washing with ice-cold PBS, freshly prepared β-galactosidase staining solution was added to each well. The plates were sealed and incubated overnight at 37 °C without CO2. The cells were analyzed under a light microscope. The results were expressed as the percentage of cells positive for blue staining in each well.
Western blot analysis
The cell lysates were separated by 10–15% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to a PVDF membrane (Millipore, Billerica, MA). The membrane was incubated with primary antibodies, including rabbit anti-ZBTB48 (Abcam, ab50588, Cambridge, United Kingdom), rabbit anti-GAPDH (Proteintech Co., 10494-1-AP, Wuhan, China), rabbit anti-P21 (Proteintech, 10355-1-AP), rabbit anti-P53 (Proteintech, 10442-1-AP), rabbit anti-CDKN2A/P16-INK4a (Bioss Co., bs-4592R, Beijing, China), rabbit anti-P14ARF (Bioss, bs0534R), rabbit anti-MDM2 (Bioss, bs-23748R), and rabbit anti-PPAR gamma (Bioss, bs-4590R) antibodies overnight at 4 °C, followed by incubation with an HRP-labeled goat or mouse anti-rabbit IgG for 1 h at room temperature. Specific proteins were detected by a high-sensitivity chemiluminescence imaging system (BioRad Laboratories, Hercules, CA, United States). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control.
Quantitative real-time PCR analysis (qRT-PCR)
Total RNA was extracted from cells using TRIzol Reagent (Takara Biochemicals, Dalian, China). cDNA was synthesized by the PrimeScript™ RT Reagent Kit (Takara) and amplified as the template by qRT-PCR with SYBR Premix Ex Taq™ (Takara) according to the manufacturer’s instructions. Relative gene expression was analyzed using the 2−∆∆Cт method. The primer sequences are listed in Additional file 1: Table S1.
In each experiment, data were acquired from three independent experiments and presented as the mean ± SEM after statistical processing. Statistical analysis was performed using SPSS 20.0 software and GraphPad 6.0 software. Comparisons were assessed by independent two-tailed Student’s t tests and one-way analysis of variance (ANOVA). The Student–Newman–Keuls multiple range test was used to determine the statistical significance of differences between means when a significant F ratio was found. *p < 0.05, **p < 0.01 and #p < 0.001 were considered to indicate different degrees statistical significance.